Purification Of Serum Immunoglobulin, Raise Of Polyclonal Antibody And Immunohistochemical Study On Lymphoid Organs In Flounder, Paralichthys Olivaceus

Flounder, Paralichthys olivaceus, a species of high commercial value and reared in costal China, has been attacked by many diseases nowadays. Vaccine may play an important role to enhance the healthy aquiculture while abuse antibiotics increases hazards to human health. In order to produce and popularize use of vaccine for fish, study fish immune system becomes compulsory. To attain more comprehensive understanding of flounder immune system, it is necessary to characterise and localise lymphoid cells within the different lymphoid organs. Use polyclonal antibody or monoclonal antibody as a tool will benefit this kind of study.This paper purified serum immunoglobulin of flounder by using Sephacryl-200 gel column chromatography after ammonium sulfate precipitation and HiTrap rProtein A Sepharose affinity chromatography. The results show that former method by which enough immunoglobulin for further study attained maybe more effective than latter. The molecular weight of flounder serum immunoglobulin is 836 kDa and that of heavy chain is 75.4 kDa, while that of light chains are 29.9 kDa and 28.2 kDa respectively.Meanwhile, polyclonal rabbit antibody against flounder serum immunoglobulin was produced. The potency determined by double immunodiflusion was 1: 32 and a few more than 1:2000 by immunodotting. The Western-blotting illuminated the antibody had immunological activity by reaction with heavy chain and light chains of flounder serum immunoglobulin and no cross reaction with that of human.The polyclonal rabbit antibody against flounder serum immunoglobulin was used as a marker for immunohistochemical detection of B cells. The present investigation reports there are two kinds of lymphoid cells which are Ig-bearing and non Ig-bearing cells existing in flounder blood. This antibody reacted with Ig-bearing lymphocytes in paraffin-embeddedsections from kidney and spleen which are main lymphoid organs in flounder. The rank of Ig-bearing lymphocytes number is head kidney, spleen and trunk kidney.In addition, hindgut has also been analysed, as studies have suggested that numerous lymphoid cells occur at this location in the lamina propria and the submucosa. The Ig-bearing cells are also found in the epithelial layer occasionally.